Summary of Work: We are able to examine the consequences of a site-specific double-strand break (DSB) in dispensable DNA within the yeast Saccharomyces cerevisiae. We showed that a persistent, long-lived DSB can lead to extended G2 arrest and eventual lethality. After screening a high-copy yeast genomic library, the SIR4 gene was identified as a component in the DSB signaling response. This, andSIR2 and SIR3, affect transcriptional silencing, chromatin organization, aging and DSB endjoining in yeast, suggesting that these genes are important in the signaling pathway. SIR genes, which repress silent mating-type gene expression, played a key role in the adaptation and lethality resulting from the DSB. Sir+ cells rapidly adapt to the persistent damage and, following a brief G2 arrest, divide and form viable microcolonies. Cells lacking Sir2, -3 or -4 exhibit prolonged G2 arrest in response to the unrepairable DSB. The sir- cells slowly adapt to the break, divide and re-arrest in the next G2. They undergo a limited number of extended G2 arrest/adaptation cycles and die as a microcolony. These SIR-mediated effects are indirect since adaptation to the G2 checkpoint was unaffected and viability was restored in the sir- strains that had also been deleted for the silent mating-type loci HML and HMR. Since deletion of RAD50 (essential for non-homologous end-joining, NHEJ) had no effect on cell viability, toleration of the DSB is not through an NHEJ pathway requiring the direct participation of Sir2, -3 or ?4. Moreover, deletion of SIR4 did not enhance the killing effects of gamma-irradiation or MMS treatment in a Rad+ or rad50D repair deficient strain. This suggests that SIR4 has no direct effect DSB repair. We conclude that toleration of an unrepaired DSB is not due to a direct effect of the SIR gene products on end-joining repair systems. Instead, the SIR effects are indirect and suggest a gene(s) that in yeast is under mating-type control plays an important role in checkpoint adaptation and toleration of a persistent DSB lesion.